Studies On Epigenetic Modification Of Cloned Bovine Embryos

Adult somatic cell nuclei can be reprogrammed into embryonic state through nuclear transfer, cytoplasm of oocyte possesses factors essential for converting differentiated somatic nuclei into pluripotent status, however, this reprogramming process is of low efficiency mainly due to incomplete somatic nucleus epigenetic remodeling. The abnormal epigenetic modifications include DNA methylation and histone acetylation that serve as monitor of chromatin structure and gene expression. In this paper, the DNA methylation and the histone acetylation status of IVF and SCNT embryos were analysed and compared, effects of epigenetic reprogramming on the development of cloned embryos were investigated.1. In this study, effects of EGF and Insulin, different sperm treatment methods, and different somatic cell co-culture systems on bovine in vitro fertilization (IVF) were investigated. The results showed that, the matured rate and cleavage rate were significantly increased after added 30 ng/mL EGF to in vitro culture medium; the swim-up method resulted in the best cleavage rate and blastocyst rate; and the blastocyst rate was significantly increased when the fertilized oocytes were cultured with bovine oviduct epidermic cells.2. In this study, effects of different activation methods and somatic cell co-culture systems on in vitro development of cloned bovine embryos were investigated. The results showed that, the cleavage rate and blastocyst rate were promoted after the fused couplets acticated with 5μmol/L ionomycin followed by 2mmol/L 6-DMAP; and the blastocyst rate was significantly increased when the cloned embryos were cultured with bovine oviduct epidermic cells.3. The DNA methylation and histone acetylation status of IVF and SCNT embryos were analysed by immunodetection method, and the differences between two kinds of embryos were investigated. The results showed that, the DNA methylation level of cloned embryos were higher than that of IVF embryos, wich was significant from 2-cell to blastocyst stage, and the precocious de novo methylation was found in cloned embryos; the histone acetylation level of cloned embryos were lower than that of IVF embryos, which became significant in 8-cell stage. The results revealed that the DNA methylation and histone acetylation status were abnormal in cloned embryos.4. The epigenetic modification status of cloned embryos were analysed after donor cells and cloned embryos were treated with 5-aza-dC, and the relationship between DNA methylation and the development of cloned embryos were investigated. The results showed that, the in vitro development potential and epigenetic modification status of cloned embryos were not changed after the donor cells or cloned embryos were treated with low concentration of 5-aza-dC (20nM, 72h); when both donor cells and cloned embryos were treated with 5-aza-dC (20nM, 72h), the blastocyst rate and blastocyst cell number were significantly increased, and the DNA methylation level of 2-cell embryos was reduced, which became significant at 8-cell stage, however, the histone acetylation level of cloned embryos was not changed. The results revealed that the DNA methylation status was important for embryo development , and reduce the DNA methylation level at 2-cell and 8-cell stages could promote the development potential of cloned embryos.5. The epigenetic modification status of cloned embryos were analysed after donor cells and cloned embryos were treated with TSA, and the relationship between histone acetylation and development of cloned embryos were investigated. The results showed that, the blastocyst rate of cloned embryos was significantly increased after donor cells treated with 50nM TSA(12h), and histone acetylation level of 2-cell embryos was increased; the in vitro development potential and epigenetic modification status of cloned embryos were not changed after the cloned embryos were treated with TSA; when both the donor cells and cloned embryos were treated with 50nM TSA(12h), the blastocyst rate was significantly increased, and the histone acetylation level of 8-cell embryos was dramatically increased and more closer to IVF embryos, however, the DNA methylation level of cloned embryos were not changed. The results revealed that hisone acetylation status of cloned embryos was important for embryo development , and increase the hisone acetylation level at 2-cell and 8-cell stages could promote the development potential of cloned embryos.6. In this study, the donor cells and cloned embryos were treated with TSA and 5-aza-dC, the following epigenetic status of cloned embryos were analysed and the effects of these reagents on development of cloned embryos were investigated. The results showed that, 5-aza-dC(20nM, 72h) combined with TSA(50nM, 12h) could significantly promoted the development potential of cloned embryos, the DNA methylation level of 2-cell and 8-cell embryos was significantly reduced while the histone acetylation level significantly increased, which became more closer to IVF embryos. The results revealed that the abnormal epigenetic status of cloned embryos could reduce the subsequent development ability, whereas change the abnormal status could promoted the subsequent development. Further studies demonstrated that the beneficial effects of TSA and 5-aza-dC on cloned embryos development were not donor cell specific.